title: Immortalized rat proximal tubule cells produce membrane bound and soluble megalin.
abstract: Megalin (gp330), a glycoprotein receptor found on renal proximal tubule cells and several other epithelial cells, is deduced to be a type I integral membrane protein, but may also exist as a cell surface form lacking a cytoplasmic domain. Furthermore, soluble megalin products have been detected in urine, and in culture medium of a rat yolk sac carcinoma cell line, combined with receptor associated protein (RAP). Permanent renal cell lines expressing megalin were unavailable until the recent description of two immortalized rat proximal tubule cell lines (IRPTC). The present study demonstrated megalin on IRPTC surface by immunofluorescence, without surface staining for RAP, which was, however, readily detected within cells. Antibodies to ectodomain megalin epitopes immunoprecipitated megalin products both from cell lysates and culture medium, whereas antibodies to cytoplasmic domain epitopes precipitated megalin only from lysates. Western blots showed two major megalin products in medium, a prominent band at approximately 200 kDa, and a fainter band above 400 kDa, slightly below intact megalin in cell lysates. Anti-receptor associated protein antibodies immunoprecipitated megalin from IRPTC lysates, but not from media. We propose that portions of megalin are spontaneously produced by IRPTC, probably either by cleavage in the ectodomain or release of forms lacking a cytoplasmic domain.
authors.affiliation: Pediatric Renal Research Laboratory, Massachusetts General Hospital, Boston 02114, USA.
authors.forename: F F
authors.initials: FF
authors.lastname: Jung
authors.forename: D R
authors.initials: DR
authors.lastname: Bachinsky
authors.forename: S S
authors.initials: SS
authors.lastname: Tang
authors.forename: G
authors.initials: G
authors.lastname: Zheng
authors.forename: D
authors.initials: D
authors.lastname: Diamant
authors.forename: L
authors.initials: L
authors.lastname: Haveran
authors.forename: R T
authors.initials: RT
authors.lastname: McCluskey
authors.forename: J R
authors.initials: JR
authors.lastname: Ingelfinger
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis; D002461:Cell Line, Transformed; D018240:Endodermal Sinus Tumor; D006023:Glycoproteins / Q000032:analysis; D026541:Heymann Nephritis Antigenic Complex; D007687:Kidney Tubules, Proximal / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D026563:LDL-Receptor Related Protein-Associated Protein; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis; D018832:Molecular Chaperones / Q000032:analysis; D051381:Rats; D011971:Receptors, Immunologic / Q000032:analysis; D011973:Receptors, LDL / Q000032:analysis; D012995:Solubility; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D006023:Glycoproteins; D026541:Heymann Nephritis Antigenic Complex; D026563:LDL-Receptor Related Protein-Associated Protein; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011971:Receptors, Immunologic; D011973:Receptors, LDL